Does M. mycetomatis biofilm have eDNA?

[laujialiang]

Good day @wwjvdsande ! I am Jia Liang, currently in my final year under the MPHARM programme at UCL. For my research project which is titled "design a molecule that can penetrate fungal biofilm", I will be mainly referring to #23 and #24.

As there are more research revolving around bacterial biofilms, I started my study on that specific area to identify if there is any similarity to fungal biofilms. The idea was if they have similar structural compositions, a molecule that can penetrate through bacterial biofilms should also be able to pierce through fungal biofilms. However, I found out that even the structures and compositions among bacterial biofilms vary between different species. Fortunately, there are a few common things regarding bacterial biofilms such as the presence of extracellular polymeric substances, water channels, proteins, and eDNA. I have also found out that eDNA is essential in biofilms as it plays a fundamental role in maintaining the structure stability of biofilms. Based on (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551309/), it is stated that through DNase treatment, the researchers were able to reduce the formation of P.aeruginosa biofilm significantly.

Then, I proceeded to read more papers regarding fungal biofilms to identify if eDNA is present in them because it was not mentioned in (https://github.com/OpenSourceMycetoma/General-Start-Here/wiki/The-Challenge-of-the-Grains). From what I gathered, Sporotrix schenckii, Candida albicans, and Aspergillus fumigatus all have eDNA and DNase treatment was proven to decrease biofilm mass in Candida albicans (https://pubmed.ncbi.nlm.nih.gov/20012895/), while it was shown that combinational treatment of biofilms with DNase plus amphotericin B and caspofungin significantly improved antifungal susceptibility towards Aspergillus fumigatus (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629765/). Thus, I believe that if eDNA is present in M. mycetomatis, similar treatment could prove to be effective. So, I am wondering if anyone could confirm if eDNA is present in M. mycetomatis?

[wwjvdsande]

@laujialiang we have grain samples in formalin. In our laboratory we have groups working with bacterial biofilms. They stain the eDNA with Propidium Iodide, that fluorescence red. We could stain it, but I need to check first if it will work on formalin fixed samples and if I can see the signal above the autofluorescence of the grains. Grains autofluoresence in both red and green. I'm not sure if there are also stains in the blue light. May be the old fashioned ethidium bromide. But that can penetrate cells if I'm correct.

[laujialiang]

@wwjvdsande Since the relationship between eDNA and biofilm EPS structural integrity is already built, wouldn't we be able to identify if eDNA is present in M. mycetomatis biofilm by measuring the effect of DNase treatment?

[wwjvdsande]

@laujialiang a true grain has never been formed in vitro, treating larvae with DNAse could also have other effects and staining of eDNA would still be necessary to demonstrate if eDNA is absent or present after DNAse treatment. We have a clumb model we can use. But how truely it represents a grain remains the question.

[laujialiang]

Hello again @wwjvdsande , first of all, thank you for your previous replies which were really helpful. I have talked to my supervisor and proceeded to read more papers.

From this paper (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629765/), I believe that they were using A. fumigatus Af293 clinical isolates instead of using a model formed in vitro, so I am wondering if we could collect grains from in vivo and working out whether eDNA is present? If that is feasible, it could rule out the other effects of DNAse on larvae.

In addition, when you mentioned the autofluorescence of the grains, have those compositions been identified yet? I have looked into (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3590280/) and (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501544/) there isn't any information about autofluorescence. I have also looked into (https://github.com/OpenSourceMycetoma/Series-1-Fenarimols/issues/23#issuecomment-551844807) as well but it was assumed that the green bits are the cement matrix.

On the other hand, I have also looked into some papers about staining of eDNA and from (https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-020-01789-5), they have used TOTO-1 signal (green) to stain eDNA, while ConA signal (orange) to stain the EPS. Through the experiment, they have concluded that the EPS and eDNA should overlap with each other given the importance of eDNA in promoting the growth and maintaining the structural integrity of the EPS. My question is would the overlap of the colours indicate anything important in M. mycetomatis grains?

[wwjvdsande]

Dear @laujialiang, in vivo grains are difficult to obtain. They can be obtained from patients but they are not in our hospital so that makes it difficult. Furthermore they will be from different strains which could also be of influence. To get them from a mouse model or a larvae model is possible. For our mouse model we currently don't run experiments and I will not get approval for an experiment like this. The larvae season just ended.

With respect to the autofluoresence. We did not identify the compounds yet. We did not isolate specific grain components and see what they are. So I don't know what the specific compound is what autofluorescences.

When staining eDNA and EPS I expect to get some overlapping results. However this might not be necessarily be directly correlated. There will be a mixture of all kind of molecules and EPS and eDNA will most likely be in the cement material but so will melanin and other proteins. A more biochemical appraoch is probably needed to unreveal all the components of the extracellular matrix.

[laujialiang]

@wwjvdsande Thank you for your clarification! Really appreciate it.